MXPA04012913A - Pasteurisation process for microbial cells and microbial oil. - Google Patents

Abstract

An improved pasteurisation protocol for pasteurising microbial cells is disclosed. The protocol has three stages, a first heating stage, a second plateau stage at which the cells are held at a (maximum and) constant temperature, and a third cooling stage. Both the heating and the cooling stages are rapid, with the temperature of the cells passing through 40 to 80 degree C in no more than 30 minutes in the heating stage. The heating rate is at least 0.5 degree C/minute and during cooling is at least -0.5 degree C/minute. The plateau maximum temperature is from 70 to 85 degree C. By plotting the pasteurisation protocol on a time (t, minutes) versus temperature (T, degree C) graph, one obtains a trapezium having an area less than 13,000 degree C minute. Not only does this result in a smaller energy input (and so a reduction in costs), but a better quality (and less oxidised) oil results having a peroxide value (POV) of less than 1.5 and an anisidine value (AnV) of less than 1.0.

Description

PASTEURIZATION PROCESS FOR MICROBIAL CELLS AND MICROBIAL OILS Field of the Invention The present invention relates to a process for pasteurizing microbial cells comprising heating the cells from 40 ° C to 70 ° C in no more than 30 minutes. The heating rate during the pasteurization process can be at least 0.5 ° C / minute. The pasteurization process can comprise three stages, namely a heating step, a plateau stage (where the cells are kept at a constant temperature) and a heating step. If the pasteurization protocol is plotted, the area under the graph time (in minutes) versus temperature (° C) is less than 13,000 ° C minute. After pasteurization, polyunsaturated fatty acids (PUFA), such as arachidonic acid, or microbial oils can be extracted from the cells. The oil may contain a low peroxide value (POV) and / or a low anisidine (AnV) index. BACKGROUND OF THE INVENTION Polyunsaturated fatty acids, or PUFAs, are found naturally and a wide variety of different PUFAs are produced by different simple cellular organisms (algae, fungi, etc.). In particular, an important PUFA is arachidonic acid (AA), which is one of the members of long-chain polyunsaturated fatty acids (LC-PUFAs). Chemically, arachidonic acid is cis -5,8,11,14 eicosatetraenoic acid (20: 4) and belongs to the family (n-6) of LC-PUFAs. Arachidonic acid is the main precursor of a wide variety of biologically active compounds known collectively as eicosanoids, a group comprising prostaglandins, thromboxanes and leukotrienes. Arachidonic acid is also one of the components of the lipid fraction of human breast milk and is essential for the optimal neurological development of children. Arachidonic acid has a wide variety of different applications including its use in formulas for infants, food and animal feed. WO-A-97/37032 (Gist-Brocades) refers to the preparation of a microbial oil containing PUFAs from the pasteurized biomass. However, it does not disclose rapid heating to a temperature at which pasteurization takes place, or cooling therefrom. In addition, it does not take into account the total amount of energy used during the pasteurization process. Both, WO-A-00/15045 and WO-A-01/67886, relate to the use of Mucous fungi for use in the preparation of edibles. The first of these documents refers to the need to perform a reduction of the RNA before including the cells in a food and suggests a heating step. The second document suggests that a heating step to reduce the RNA content can be avoided by leaving the fungal cells stored in the container and allowing them to "mature". The application of the international patent No. PCT / EP01 / 08902 refers to the process for preparing blends of oils by combining oils containing crude PUFAs -6 with crude co-3 to produce a mixture of oils and then to the purification of the mixture of crude oils. Processes that involve the heating of biomass or microbial cells are known. From WO-A-97/37032, it is also known that microbial cells can be pasteurized prior to the extraction of a PUFA in the form of an oil. However, the present applicants have found that a new pasteurization process can improve the quality of an oil that can be extracted from the pasteurized cells. In particular, the resulting oil may be oxidized less, or be less oxidizable, and may have a low peroxide index (POV) and / or a low anisidine index (AnV). In addition, applicants have found that this new pasteurization process is more efficient because it requires less energy. Thus, the process is advantageous because not only can it improve the quality of the oil, but it can also reduce costs since it requires less energy. BRIEF DESCRIPTION OF THE FIGURES Figure 1 is a graph of temperature (° C) as a function of time (minutes) for three pasteurization protocols (A and C are within the invention, B is provided for comparison). Figure 2 is a graph of temperature (° C) as a function of time (minutes) for pasteurizations at three different plateau temperatures (40, 70 and 85 ° C). Figures 2 and 4 are graphs of AnV (and PUV of Fig.3) as a function of time (hours). Figure 5 is a plot of POV (meq / kg) and AnV as a function of temperature for pasteurizations at two different (plateau / stay) times (8 and 300 seconds). Figures 6 and 7 are temperature graphs (° C) as a function of time (seconds) for two different (plateau / permanence) times (8 seconds for Figure 6, 5 minutes for Figure 7) at five different temperatures (60 , 80, 100, 120 and 140 ° C).

Description of the Invention The present invention, therefore, provides an improved microbial cell pasteurization process. As the pasteurization process of the invention requires less energy it can allow a better quality of a product. Thus, a first aspect of the present invention relates to a process for the pasteurization of microbial cells, the process comprises heating the cells (at temperatures they comprise) from 40 ° C to (60 ° C or 70 ° C in no more than 30 minutes or the heating of the cells at a speed of at least 0.5 ° C / minute.Therefore, this aspect provides a rapid heating of the microbial cells during pasteurization and such a high heating rate is not disclosed in the matter While the matter provides pasteurization temperatures, there is no appreciation or discussion of the heating rate, or that this parameter could be important and that a relatively fast speed can provide benefits.Indeed, the high heating rates are intuitively against, as it can be expected to cause oxidation or, on the other hand, degradation of PUFAs or oils that can be extracted from the cells the .

A second aspect of the second invention relates to a process for the pasteurization of microbial cells, the process encompassing a pasteurization protocol comprising (at least) three stages. They are, namely: a (first) heating step, a (second) stage plateau (in which the microbial cells are left at a desired temperature or where the cells are kept at a constant and / or maximum temperature), and a (third) stage of enfilation. This aspect of the invention is referred to as a three stage pasteurization protocol. If this protocol is taken to a graph of time versus temperature a trapezoid may result. A third aspect of the invention relates to a pasteurization process for microbial cells, the process comprises the use of a pasteurization protocol such that the area included in the graph of time (minutes) versus temperature (° C) is less than 13,000 ° C. minute. The area below the graph of time versus temperature provides the amount of energy consumed in heating the cells during the pasteurization process. It has been found that rapid heating and / or rapid cooling of the cells (corresponding to the first and the third stage of the second aspect, respectively) can provide advantages, such as a better quality oil. In addition, the amount of energy required for the pasteurization process can be reduced compared to pasteurization processes described in the art. This third aspect concerns the input energy required for the pasteurization process. A fourth aspect of the invention relates to a process for pasteurizing microbial cells, the process comprises (heating the cells and thus) maintaining the cells at an elevated temperature (T, ° C) for a time (t, minutes) , for example, in a plateau stage, where the product tT (ie the multiplication of the time and temperature parameters, for example, during the plateau stage) is from 140 to 100,800 ° C. minute. As can be appreciated, this fourth aspect is similar to the second aspect in that it contains a plateau stage. The cells here can be left at a constant or maximum temperature. The product tT can thus represent the area below the time versus temperature graph for this plateau stage. First Aspect - Rapid Heating In this aspect the cells are heated to those temperatures at which the cells pass through or from 40 ° C to 70 ° C in no more than 30 minutes (as well as no more than 15 minutes). Preferably the time taken to go from 40 to 70 ° C should be no more than 40 to 50 minutes.

Alternatively or in addition, the cells are heated at a speed of at least 0.5 ° C / minute. Of course, the microbial cells can be (or be heated) at a temperature below 40 ° C. For example, the cells can start at room temperature or in the room. The cells may be at the fermentation temperature, such as at 30 ° ± 5 ° C. Thus, cells can be from 20 to 40 ° C, as well as from 23 to 27 ° C (or from 25 or 29 to 32 or 37 ° C) when heating (pasteurization) begins. In some cases the microbial cells can be cooled, for example, after the end of a fermentation. Thus, the cells can have an (initial) temperature of 5 to 10 ° C, as well as, of 7 to 9 ° C when the heating begins. The microbial cells can therefore be heated until their temperatures are below (60 o) 70 ° C. Thus, this may not be the final temperature of the microbial cells during pasteurization. In effect, the cells can be heated to a temperature below (60 o) 70 ° C. The temperature can be raised to a temperature of 70 to 90, 110 or 130 ° C, as well as, from 75 to 87 ° C and optimally rises from 78 to 84 ° C. The maximum temperature during pasteurization can be within these ranges, but for some embodiments can reach up to 100, 120 or 140 ° C. Preferably the cells are left or maintained at that (maximum) temperature. From now on it can be recognized that the cells can be heated to a lower temperature, or start, from 40 ° C to a temperature of 70 ° C or higher. The range of 40 to 70 ° C can provide a "strong shot" in extending the heating / temperature range for which a time (and thus a range) can be specified (and thus calculated). It can be calculated that the heating (from 40 to 70 ° C in 30 minutes) is at a speed of 1 ° C / minute. However, if necessary, the speed can be lowered slightly and in the first aspect rapid heating means a heating rate greater than 0.5 ° C / minute. Preferably, the speed is at least 0.6; 1.0 or equal to 1.5 ° C / minute. However, particularly rapid heating rates are contemplated, depending on the equipment and the volume or weight of the microbial cells that must be heated. Heating speeds that exceed 2.0 ° C or are equal to 2.5 ° C / minute are thus within the invention. Particularly high heating speeds can be achieved by using special equipment. This can reach a high temperature in a short period of time and thus any oxidation or damage of the PUFA or microbial oil that can be subsequently isolated can be minimized. Thus, the heating must be carried out up to a maximum temperature of up to 140, 150 or equal to 160 ° C. Preferably, the heating can reach a temperature range within 100 to 180 ° C, such as 120 to 160 ° C, preferably 130 to 150 ° C. These temperatures can be achieved quickly using fast heaters, for example, within less than one minute (30 seconds). Each temperature can be reached within 20, 30, 40 or 50 seconds, or it can rise to 150, 175, 200, 225 or 250 seconds. However, each temperature can be reached in small times such as 2, 4, 6, 8 or 10 seconds, using, for example, infusion heaters or relatively small samples. Thus, heating speeds up to 50, 100, 150 or even 200 ° C per minute are achieved. Thus slightly lower heating rates of 5 or 10 at 50 or 60 ° C per minute are possible, as well as from 15 to 45 ° C per minute. This rapid heating during rapid pasteurization has to be found not only to be more effective and require less energy, but to be at least one factor responsible for obtaining a better quality microbial oil (once extracted from the cells after the pasteurization).

Second Aspect- Pasteurization Protocol in Three Stages The first stage can be a warm-up stage. In effect this corresponds to the rapid heating described in the first aspect of the invention and in this way, all the features and characteristics of the first aspect are applied mutatis mutandis to the first stage of the second aspect. The second stage corresponds to the cells when they are on a plateau (temperature). The cells can be stored at a particular and desired temperature (more or less 1 or 2, or equal to 10 ° C) for the desired time. The cells can thus be maintained at a constant temperature. In the plateau stage this temperature (or range of temperatures) is preferably the maximum temperature reached during the pasteurization protocol. The temperature of the plateau stage (and / or the maximum temperature during pasteurization) is preferably at least 70 ° C. It can be less than 90 or 100 ° C, more convenient from 70 to 85 ° C, as well as from 70 to 77 ° C. Alternatively, it can be between 80-160 ° C, as well as, between 100-140 ° C. The length of time of the plateau stage, or the desired one at which the cells are conserved, or at the maximum temperature, should be from 5 seconds to 90 minutes, as well as from 10 to 80 minutes, for example, from 20 up to 70 minutes. This time is optimal from 40 or 50 to 60 or 70 minutes, as well as, from 45 to 65 minutes and more advantageous from 55 to 63 minutes. Also particularly short times are possible, for e. from 8 seconds to 5 minutes. The third stage is a cooling stage. Preferably, the cells are cooled to a temperature that is the same, or is within the ranges mentioned at the beginning of the heating (or first stage). Preferably, the microbial cells are heated and / or cooled linearly (in the first and / or in the third stage, according to convenience), this is so that the cooling or heating profiles appear (approximately) as a straight line when plotting Time versus temperature. The cells can be kept cold, or they can be actively cooled, for example using a heat exchanger and / or a cooling substance, for example, at room temperature (lowering it), or at room temperature, or lower. Preferably the cooling rate is at least 0.4; 0.6; 1.0 or 1.5 ° C / minute. These values represent cooling rates achieved when the cells are allowed to cool on their own. However, faster cooling speeds are possible if a coolant is used. Thus, cooling speeds of at least 2.0; 2.5; 3.0 or up to 3.5 ° C / minute are accessible. However, higher cooling rates are possible, such as below 5 ° C per minute, eg. from 7 or 10 to 50 or 60 ° C per minute, preferably from 15 to 45 ° C per minute. The preferred heating and / or cooling rates are maintained at least at 10, 20 or 30 ° C, although in some embodiments these can be achieved in a range of at least 40 or 50 ° C. It could be recognized that a rapid heating stage and a rapid cooling stage can reduce the amount of energy used in pasteurization. This result not only can achieve lower costs, but also may not adversely affect the quality (of the eventual) microbial oil, in effect, it seems to have beneficial effects on the oil. Third Aspect-Area. Below the Time versus Temperature Graph (Energy Delivery) From the second aspect, it will be evident that, if with the protocol of the invention a time versus temperature graph is plotted, a trapezoidal figure is obtained. The first (heating) and the third stages (cooling) can each form a triangular shape, while the middle or second stage (plateau) (the object of the fourth aspect) is (usually) rectangular. The area below the time versus temperature graph represents the amount of energy consumed in the system. By dividing the pasteurization protocol into three parts, you can calculate the area of the graph, and thus the energy consumed. In the third aspect, the area below the graph of time (in minutes) versus temperature (in ° C) is less than 13,000 ° C. minute. However, lower amounts can be achieved than this and values below 11,000; 10,000; 9,000; 8,000 or equal to 1,000 ° C. minute. In preferred aspects of this invention, these values may be no greater than 7,000; 6,000 or 800 ° C. minute. In the referred chart, time is represented on the x axis (or horizontal axis or abscissa) and 0 ° C represents the origin. The temperature can be plotted on the y axis (or vertical or ordered axis) and 0 ° C represents the origin. Once the cells have been heated to their pasteurization temperature, they can then be cooled (or cooled). The cells are usually cooled to room temperature or room temperature, or at least to a temperature below 30 ° C. There is therefore a time not only for the cells to warm from 30 to 60 ° C, but also a time for the cells to cool from 60 ° C down to 30 ° C. These two times can be summarized to provide a Combined heating and cooling time of 30-60 at 30 ° C. Preferably, this combination is only less than 150 minutes, such as less than 120 or 100 minutes. However, with smaller samples longer times can be achieved and the combined time (30 to 60 and lower to 30 ° C) can be less than 70, 50 or equal to 30 minutes. Fifth Aspect- Pasteurization Protocol with Plateau Stage This protocol can be in accordance with the second aspect, where there is (eg first) a heating stage and (eg second) a cooling stage, in the middle (eg second or middle or intermediate) a plateau stage. However, it is not essential and other pasteurization protocols can be contemplated. The fourth aspect relates preferred features of this stage of the plateau. All the features and characteristics of the second (and other) aspect are applied mutatis mutandis to this fourth aspect. The cells are maintained or left at a particular desired temperature (more or less 1, 2, 5 or equal to 10 ° C) at a temperature (T, ° C) for a time (t, minutes). These two parameters can multiply together giving the product tT. This is convenient from 140 or 280 to 50,000 or 100,800 ° C. minute. Preferably this product is from 500, 1,000, 2,000 or 3,000 or equal to 6,000 to 10,000, 18,000 or 25,000 ° C. minute Optimally, the product tT is from 2,000 to 6,000, as well as, from 3,000 to 5,000, optimally from 4,000 to 4,500 ° C. minute. In some embodiments, the product tT is from 13 to 900, as well as from 100 or 200 to 700 or 800, optimally from 300 to 400 to 600 or 700 ° C. minute. Thus in a manner similar to the third aspect, you may realize that the product tT represents the area under the graph of time versus temperature of the cells when kept at an elevated temperature. Thus, the multiplying factor tT is, in effect, the area under the graph during the plateau stage (but not during the heating or cooling stages). Extraction of a PUFA A fifth aspect of the present invention relates to a process for obtaining a PUFA from microbial cells, the process comprises the pasteurization of the cells according to any (first, second, third or fourth) of the aspects of the invention, as previously described, and extracting and / or isolating a PUFA from the pasteurized cells. A sixth aspect of the present invention relates to a microbial oil which may contain at least 40% arachidonic acid (AA) and / or may have a triglyceride content of at least 90%. The oil may have a POV less than 2.5; 1.5; 0.8; 0.6 or equal to 0.5 and / or an AnV less than 1.0. The oil is prepared by the process of the fifth aspect. Polyunsaturated fatty acids (PUFAs) and microbial oils The PUFA can be a simple PUFA or two or more different PUFAs. The or each PUFA can be of the n-3 or n-6 family. Preferably it is a C18, C20 or C22 PUFA. It can be a PUFA with at least 18 carbon atoms and / or at least 3 or 4 double bonds. The PUFA can be provided in the form of a free fatty acid, a salt, as a fatty acid ester (eg methyl or ethyl ester), as a phospholipid and / or in the form of a mono-, di- or triglyceride. Convenient PUFAs (n-3 and n-6) include: docosahexaenoic acid (DHA, 22: 6 n-3) available in algae or fungi such as Crythecodinium (dinoflagellate) or the Thraustochytrium (fungus); y-linolenic acid (GLA, 18: 3 n-6); a-linolenic acid (ALA, 18: 3 n-3); Conjugated linoleic acid (octadecadienoic acid, CLA); dihomo- acid? -linoleic (DGLA, 20: 3 n-6); arachidonic acid (AA, 20: 4 n-6) eicosapentaenoic acid (???, 20: 5 n-3).

Preferred PUFAs include arachidonic acid (??), docosahexaenoic acid (DHA), eicosapentaenoic acid (EPA) and / or? -linoleic acid (GLA). In particular, the preferred one is AA. The PUFA can be produced by the pasteurized cells in the process of the invention, such as a microbial cell. They can be bacterial cells, algae, fungi or yeast. Fungi are preferred, especially of the order Mocorales, for example, Mortierella, Phycomyces, Blakeslea, Aspercillus, Thraustochytrium, Pythium, or Entomophthora. The preferred source of AA is Morteriella alpina,, Blakeslea trispora, Aspergillus terreus and Pythium insidiosum. The algae can be dinoflagellate and / or even Porphyridium, Nitszchia, or Crypthecodinium (eg Crypthecodiniurn chonií). Yeasts include those of the genus Pichia or Saccharomyces such as Pichia ciferii. The bacteria can be of the genus Propionibacterium. The microbial oil can be liquid (at room temperature). It is preferred that most PUFAs are in the form of triglycerides. Thus, it is preferred that at least 50%, such as at least 60% or optimally at least 70% of the PUFAs are as triglycerides. However, the amount of triglycerides in the oil can be greater, at least 85%, preferably at least 90%, optimally at least 95 or 98%. In these triglycerides preferably at least 40%, such as at least 50% and optimally at least 60% of the PUFAs are in the a position of the glycerol (present in the triglyceride backbone) known as position 1 or 3. Preferred that at least 20%, such as at least 30%, optimally at least 40% of the PUFAs are in position ß (2). The microbial oil may comprise at least 10, 35, 40 or 45% or more of a desired PUFA such as arachidonic acid. It can contain at least 90% triglycerides. Preferably the microbial oils have a triglyceride content of 90 to 100%, at least 96%, preferably at least 98%, more preferably at least 99% and optimally above 99.5%. The microbial oils will typically contain an amount of eicosapentaenoic acid (EPA) of less than 5%, preferably less than 1% and more preferably less than 0.5%. The oil may have less than 5%, less than 2%, less than 1% of each of the PUFAs of C20, C2o-. 3, C22: or? / C20:. The content of free fatty acids (FFA) must be less than or equal to 0.4; 0.2 or 0.1. The oil may or may not have GLA and / or DGLA. The microbial oil can be a crude oil. It can be extracted from the cells using a solvent such as supercritical carbon dioxide, hexane or isopropanol.

Pasteurization process Pasteurization generally takes place after the fermentation has finished. In a preferred embodiment, pasteurization can end with fermentation because heating during pasteurization will kill the cells. Pasteurization, therefore, can be carried out in the fermentation broth (or the cells in an aqueous medium), although it can be carried out in the microbial biomass obtained from both. In the first case, the pasteurization takes place while the microbial cells are still in the thermenter. Preferably the pasteurization takes place before any further processing of the microbial cells, for example, granulation (eg extrusion) comminuted or kneaded. Preferably the pasteurization protocol is sufficient to inhibit or inactivate one or more enzymes, for example, a lipase, which can adversely affect or degrade a PUFA or a microbial oil. Once the fermentation has finished, the fermentation broth can be filtered, or on the other hand, treated to remove the water or the aqueous liquid. After removing the water, a "cake" of biomass can be obtained. If the pasteurization was not performed, then the drained cells (or biomass cake) can be pasteurized.

Process for Removing PUFAs PUFAs (or the microbial oil that usually contains PUFAs) can be extracted from microbial (pasteurized) cells. Preferably, they are extracted from the granules (eg, dried) that contain the cells (eg extruded). The extraction can be done using a solvent. Preferably a non-polar solvent is used, for example an alkane C, preferably a C 2 -alkane, for example, hexane. Carbon dioxide can also be used (in liquid form, for example in the supercritical state). Preferably, the solvent is allowed to filter on the dried granules. In WO-A-97/37032, suitable techniques for granulation and extrusion of microorganisms and subsequent extraction of microbial PUFAs contained in the oil are described. The solvent allows obtaining an oil containing crude PUFAs. This oil can be used in this state, without further processing, or it can be subjected to one or more refining steps. However, a crude oil is generally one that contains a solvent, such as that used to extract oil (eg hexane or an alcohol such as isopropanol) or that has not been subjected to one (or preferably none) of the following steps of refinement. In the applications of International Patent No. PCT / EP01 / 08902 appropriate refining protocols are described (the contents of this document and that of all others described herein are incorporated by reference). For example, the oil may be subjected to one or more of the refining steps, which may include acid treatment or degumming, alkali treatment or removal of free fatty acids, bleaching or pigment removal, filtration, winterization (or cooling, for example to eliminate saturated triglycerides), deodorization (or elimination of free fatty acids) and / or polishing (or elimination of insoluble substances in oil). All of these refining steps are described in great detail in PCT / EP01 / 08902 and muta isis mutandis can be applied in the steps described in the present invention. The resulting oil is particularly suitable for nutritional purposes and can be added to food (human) or food (animal). Examples include milk, formulas for infants, healthy drinks, bread and animal feed. Microbial cells The microbial cells (or microorganisms) used in the present invention can be any of those described above especially in the PUFAs and microbial oils section. They must comprise, or be capable of producing, a PUFA or a microbial oil and suitable for the extraction or isolation of the PUFA oil from the cells. They can be filamentous forms, such as fungi and bacteria, or simple cells such as yeasts, algae and fungi. The cells can comprise microorganisms that are yeasts, algae and bacteria. The preferred fungi are of the order Mucorales for example, the fungus can be of genus Mortierella, Phycomyces, Blakeslea or Aspergillus. Fungi of the species Morteriella alpina, Blakeslea trispora and Aspergillus terreus are preferred. With respect to yeasts they are preferably of the genus Pichia (such as the species Pichia ciferrii) or Saecharomyees. The bacteria can be of the genus Propionibacterium. If the cells come from algae, a dinoflagellate is preferred and / or it belongs to the genus Crypthecodinium. Algae of the species Crypthecodinium cohnii are preferred. Heating This can be done by heating (of the cells) directly or indirectly. If the heating is direct, it can be done by vapor passage inside the fermenter. An indirect method can use a medium as heat exchangers, either through the wall of the fermenter or through heating coils or an external heat exchanger such as a heating plate. Generally, pasteurization takes place in the fermenter vessel where fermentation occurs. Nevertheless, for some organisms (such as bacteria) it is preferred to first remove the cells from the container and then pasteurize. Pasteurization takes place before other processing of organisms, for example, drying or granulation. Pasteurization will usually kill most, or all microorganisms. After pasteurization at least 95%, 96% or up to 98% of the microorganisms have to be dead, that is, they are not alive. Acidification In some cases it is desired to reduce the risk of growth of the pasteurized cells. One possibility is to acidify the cells with an appropriate acid. Thus, in order to prevent the growth of external bacterial species, adjustment of the pH of the cells in a range of 3 to 4 may be desired. However, a wide pH range may be employed depending on the cell, and thus the pH may be Adjust from 2 to 5, optimally to a range of approximately 3.3 to 3.7.

The acidification of the cells can be carried out before pasteurization. However, it is preferable to do it later. The pH can be adjusted by any appropriate means or with any suitable acid. Preferably it is carried out using phosphoric acid, such as 85% or phosphoric acid diluted 55% or 33%. peroxide index (POV) Preferably the POV of a microbial oil is from 4 to 8 or 12, especially for crude oils. However, the POV could be no higher than 3.0; 2.5 or 2.0. However, lower values of POV can be obtained using the process of the invention, and those values can be less than 1.5 or less than 1.0. POV values less than 0.8 or 0.6 and less than 0.4 can be obtained. The values (in the realizations) are within a range of 1.3 (or 0.8) to 0.4. The unit (for POV) is usually meq / kg. anisidine index (AnV) This value can give a measure of the aldehyde content. Preferably the anisidine number of a microbial oil is from 5, 6, 7 or 10 to 15, 20 or 25, especially for a crude oil. The appropriate AnV is no more than 20, for example no more than 15. It can be no more than 10 or no more than 5. Preferably the POV and / or AnV values refer to crude oils rather than refined oils. The values of AnV (in preferred experiments) are within a range of 15 to 5, optionally 12 to 7. Crude versus refined oils The following are some differences between these two oils. Each crude or refined oil may have one or more traces of the following Table for crude or refined oils. A crude oil will usually contain an antioxidant (eg, tocopherol, ascorbyl palmitate).

Preferred Substance Crude oil Refined oil (for crude) Insaparable < 3.5% (w / w) 2.5% (w / w) 1.8% (w / w) Solvent (ex. Exano) < 2000 ppm 100-2000 ppm Not detectable or < lppm Phospholipids% 2-3.5 0.05 Free fatty acids, < 1% 0.20% 0.08% as oleic POV < 10 meq / kg 6 meq / kg 1.4 meq / kg Not soluble < 0.5% 0.10% Phosphorous < 1000 mg / kg 5 mg / kg Silicones < 500 ppm 100 ppm 2 ppm Arsenic < 0.5 mg / kg < 0.04 mg / kg < 0.5 mg / kg Cadmium < 0.2 mg / kg < 0.02 mg / kg < 0.1 mg / kg Mercury < 0.04 mg / kg < 0.4 mg / kg < 0.0 mg / kg (continued) Conveniently, the crude oil in the present invention may have one or more of the following features: (a) a content of unsaponifiables from 2.0 to 3.5% (W / W); (b) a solvent content (eg, hexane) of 10, 50 or 100 ppm at 1,000, 1,500 or 2,000 ppm; (c) a content of free fatty acids from 0.1 or 2.0% to 1.0%, eg.0.2-0.6 or 0.3-0.5%; (d) a POV value from 2,3,4 or 6 to 10; (e) a phosphorus content of at least 2.3 or 5 mg / kg; (f) a silicone content of 50 or 100 ppm; I (g) a water content less than 1% or from 0.5 to 1 or 2%. Uses of oils and PUFAs A sixth aspect of the invention relates to a composition containing the oil of the fifth aspect and where there is an appropriate or more (additional) substances.

The composition may be an edible and / or a food supplement for animals or humans. In embodiments of the invention that are for human consumption the oils may become suitable for human consumption, typically by refining or purifying the oil obtained from the microbes. The composition can be a formula for infants or edible (human). In this case, the composition of the formula must be adjusted so that it has a quantity of lipids or PUFAs similar to breast milk.

This may involve mixing the microbial oil of the invention with other oils in order to achieve an appropriate composition. The composition can be a food composition or animal or marine supplement. Each food or supplement can be obtained from any farm animal, in particular sheep, cattle and poultry. In addition, food or supplements can be obtained from marine organisms such as fish and molluscs. The composition can thus include one or more food substances or ingredients of each animal. The oil of the invention can be sold directly as oil and contained in an appropriate container, generally an aluminum container covered in its interior by an epoxyphenolic lacquer and gassed with nitrogen. The oil may contain one or more antioxidants (eg, tocopherol, vitamin E, palmitate) each, for example, at a concentration of 50 to 800 ppm, such as 100 to 700 ppm. Appropriate compositions may include pharmaceutical and veterinary compositions, e.g. oral administration or cosmetic compositions. The oil may be administered as such or may be encapsulated, for example in a shell and may thus be in the form of a capsule. The caps or capsules may contain gelatin and / or glycerol. The composition may contain other ingredients, for example flavorings (eg lemon or lime flavor) or a vehicle or excipient accepted in pharmaceutical and veterinary. The features and characteristics of one aspect of the invention are applicable rnutatis mutandis to another aspect. The invention will be described by way of examples with reference to the following Examples, which are provided by means of illustrations and are not intended to limit the scope. Example 1 Oxidation during the production of a microbial oil containing PUFAs is caused by enzymatic activity. . Pasteurization is considered as a method of stabilizing oxidation during the processing of microbial cells to obtain an oil. The extent of the stabilization was found to depend on the conditions of the pasteurization. A number of experiments were conducted to determine which pasteurization conditions may affect the oxidation levels and, in particular, the peroxide value (POV) of the oil. The peroxide indices were determined using the standard protocol detailed in AOCS: Cd8-53. The experiments follow the following protocol: fermentation; storage; pasteurization; extraction (microbial oil); oil analysis. The fungus Mortierella alpina was cultivated in a termendor. The fermentation takes approximately 148 hours. M. Alpina produces the PUFA called arachidonic acid (AA). The biomass was removed from the fermenter and stored (at a temperature below -18 ° C). Samples of the biomass of M. Alpina were removed from the fermentation broth when they were still inside the fermenter and immediately frozen. Several pasteurization protocols were tested. The pasteurization was carried out at three different temperatures, namely 40, 70 and 85 ° C. The protocol followed a process in three stages, with a first stage of rapid heating followed by a plateau (a second or a half stage) at a temperature desired which is the maximum temperature used. Continues a stage (third) of rapid cooling. Different samples of the biomass are subjected to an average stage (plateau) of three different temperatures, namely one, two and 24 hours.

After pasteurization, the microbial oil is obtained using a wet extraction technique. This sample of biomass was filtered, squeezed (under pressure) and the oil extracted. The microbial oil is then analyzed, above all for the peroxide index (POV) using an AOCS method. The AA content of some samples was determined. The analyzes showed that the obtained microbial oil had approximately 420 g of AA per kg. Detailed Protocol: Fermentation and Extraction of Samples. One liter of the fermentation broth was removed from the fermenter vessel and filtered (Seitz two-liter filter, F-FA10). The resulting cake was then washed with 600 ml of distilled water. The wet cake is dried by airflow for one minute and then pressed (using a HAFICO ™ apparatus, dyeing press, C-0A021, 300-400 atm) at 400 bar. The wet extrudate was then used to extract a microbial oil with 500 ml of hexane (Merck) at room temperature (20 to 25 ° C) for one hour using Ultra Turrax ™ equipment. The hexane was then decanted. The remaining cake was then washed with 250 ml of fresh hexane (with stirring for 30 minutes) at room temperature. The hexane was decanted and added to the previous hexane extract. The extract was then filtered using a glass filter in combination with a GFA glass filter. The hexane was then evaporated from the clear extract at 50 ° C for approximately 15 minutes using a Rotavapor ™ equipment. The oil was then transferred to sealed containers and each sample was then gassed with nitrogen for 30 seconds. The sample was then closed and stored at -18 ° C. Pasteurization Protocols Three different protocols were tested (A, B and C). Each one consists of three stages, a first stage of heating, a second stage plateau (at a maximum temperature) and a third stage of cooling. Next, Table 1 shows the protocols of the three pasteurization profiles. t o Time Temp Stage Change Time Area Speed Time Times Low Area Table 1 (t, min.) (T, ° C) at temp in per stage under Cal / Enf to pass Comb 40- graf. t vs. Time (t) stage (° C) (min) of the profile (° C / min) of 40-70 ° C 70-40 ° C T (° C.min) (min) (min) ("C.min) Profile A 0 25 75 70 heat 45 t heat = 75 1687.5 0.6 50 7575 135 70 pasteur. 0 t past = 60 4200 0 210 25 cold 45 t cold = 65 1687.5 0.6 50 100 Profile B 0 25 (out of 102 72 heat 48 t heat = 102 4896 0.46 65.11 13968 inv for 162 72 pasteur. = 60 4320 0 comp) 360 28 cold 48 t cold = 198 4752 0.22 135 200.11 Profile C 0 7 25 70 heat 63 t heat = 25 787.5 2.52 11.9 5607.5 85 70 pasteur. 0 t past = 60 4200 0 105 8 cold 62 t cold = 20 620 3.1 9, -8 21.58 The three pasteurization profiles A, B and C are also shown in Figure 1. It can be seen that the area under the curve of the temperature graph (T °, C) versus time (t, minutes) can be calculated for each of the three steps of each profile and then add them to calculate the area under the graph for each of the three profiles. These calculations are also shown in the previous Table 1. The peroxide index (POV) was determined for the oils that resulted from the extraction of the cells followed by the three pasteurization protocols A, B and C.

The POV of the extracted oil were 8.7; 14.3 and 2.4; respectively. Profile B had a slow heating rate and a slow cooling speed and is presented for comparison only. He has the greatest POV of 14.3. In contrast, profiles A and C are both within the invention. Profile A has faster heating and cooling speeds in the first and third stages than profile B. Preferably, in the invention the heating and cooling rates are at least as fast as those shown in profile A. Profile A has a POV of 8.7. However, the best results were obtained using profile C, which had a POV of only 2.4. As can be seen in Figure 1, this had a very rapid heating stage and a rapid (third) cooling stage. Example 2 Experiments similar to Example 1 were carried out except that in this case the pasteurization temperature was modified more broadly, namely at 40 ° C (for comparison), 70 ° C and 85 ° C. The temperature profile (° C) vs. time (minutes) is shown in Figure 2 and Table 2. The profile was essentially the same for all samples, but of course with an appropriate extension of the pasteurization plateau (from one hour to 4 or 24 hours). Table 2 Table 2 (continued) Samples from two different fermentations (both from M. Alpina) were analyzed. Samples Nos. 11 to 20. Table 3 has a slightly longer fermentation where approximately 2m3 of broth was transferred to an inoculant fermenter and the fermentation was extended for 48 hours without any future added glucose. After pasteurization, the samples were processed starting with a pressure filtration of approximately 1 bar of nitrogen. The resulting cake was then washed with distilled water (approximately 0.6 of the initial volume of broth). The water was removed using a press with a piston at a pressure of 300 to 400 bar. Then, 500 ml of fresh hexane was added and mixed using Ultra-turrax equipment for one minute. The extraction takes place for about one hour at room temperature. After filtration, the resulting cake was washed with 250 ml of fresh hexane and the resulting solvent was evaporated under vacuum from 60 ° C to 70 ° C. Then he gassed with nitrogen and kept at -18 ° C. The results are shown in Table 3, which includes a first and a second measurement of the peroxide value and an average of these two values, as well as the anisidine indexes (AnV). The reduction of POV and AnV are also shown in Figures 3 and 4 (for shorter and longer fermentations, respectively). Table 3 Table 3. (continued) From the results it can be seen that without pasteurization the POV was 5.6 or 5.7. Pasteurization at 40 ° C may reduce the POV, but relatively longer times (such as 24 hours) at the pasteurization temperature are required in order to reduce the POV to an acceptable value of 2.1. The highest temperatures are considered the most successful. For example, pasteurization for only 1 hour at 70 ° C gives a POV of 2.2 when compared to a POV of 2.1 for 24 hours at 40 ° C. The best values were obtained at the highest temperatures, with 85 ° C for 1 hour giving a POV value of only 1.2. (These figures are cited for shorter fermentations, although similar results may be found with cells growing in longer fermentations). Figures 3 and 4 thus show graphically how the values of POV and AnV change with respect to the different pasteurization times. As expected, the longer pasteurization times give the lowest values of POV and AnV. However, the use of relatively high temperatures during pasteurization is of more importance. A marked decrease in AnV and POV was found when the pasteurization temperature (Tpast) was increased to 70 ° C, and even lower values were found at 85 ° C. (The three upper lines indicated with crosses, full circles and asterisks show the values of AnV, while the three lower lines indicated with diamonds, squares and triangles give the values of POV). Table 4 shows the calculated product tT (in ° C minute) for nine different pasteurization protocols (three different plateau temperatures and for three different times). In effect, this product represents the area under the graph (time, t, minutes vs. temperature, T, ° C) for the plateau stage (after the heating stage, but before the cooling stage).

Table 4 EXAMPLE 3 Future pasteurization tests were performed using fermentation broths following large scale production fermentations using the M. alpina fungus, as exemplified above. An unpasteurized broth (800 liters) was transported and stored at 4 ° C. The broth was then transferred to a 700 liter vessel with agitation and 10 different pasteurization protocols were performed. First, pasteurization was carried out at five different (maximum) temperatures, namely 140, 120, 100, 80 and 60 ° C in a permanent time (plateau) for 8 seconds (at maximum temperature). Second, pasteurization was performed at 140, 120, 100, 80 and 60 ° C at a permanent time (plateau) at maximum temperature for 300 seconds.

Samples were taken (2 liters) and directly frozen at -18 ° C. Sterile samples (200 ml) were taken and frozen, and the crude AA oil was recovered from the samples using the following protocol. A sample of the fermentation broth (1.7 liters) was filtered at 1 bar of N2. The cake was washed with 0.6 volumes of distilled water and squeezed for approximately 5 minutes at 400 kg / cm2. Then, n-hexane was added to the wet cake and crumbled using Ultra Turrax equipment at 24,000 rpm. The oil was extracted at room temperature (approximately 21 ° C) for approximately 110 minutes. The suspension was vacuum filtered using a GF / A Whatman media filter. The cake was washed with 250 ml of fresh hexane. The hexane was evaporated for 15 minutes in a water bath at a temperature of about 60 to 70 ° C. The resulting oil was then transferred to an airtight container, which was gassed with nitrogen for 30 seconds and then closed and stored at -18 ° C before being analyzed. Figures 5, 6 and 7 provide the following data for analysis. Figures 6 and 7 show the time profiles as a function of temperature for the two sets of experiments, the first with a plateau time (permanence) of 8 seconds, and the second with a plateau time of 5 minutes, each of the 5 set temperatures, respectively. As can be seen from the graph, the horizontal middle line (representing 8 seconds or 5 minutes) shows the plateau stage. Figure 5 shows the values of POV and AnV that result from all 10 pasteurization regimes. As can be seen, the lowest values of POV were obtained with the increase of the high temperatures and the longer residence time (5 minutes) provides the lowest value of POV.

Claims (4)

NOVELTY OF THE INVENTION Having described the present invention, it is considered as a novelty and, therefore, the content of the following is claimed as property: CLAIMS

1. A process for pasteurizing microbial cells, characterized in that the process comprises heating the cells at a temperature comprised from 40 ° C to 70 ° C and not more than 30 minutes or at a speed greater than 0.5 ° C / minute.

2. A process for pasteurizing microbial cells characterized in that it comprises three stages, namely a heating stage (first), a (second) plateau stage (in which the cells are kept at a constant temperature) and a cooling stage (third).

3. A process for pasteurizing microbial cells, characterized in that the process comprises heating the cells using a pasteurization protocol such that the area below the graph of time (minutes) versus temperature (° C) is less than 13,000 ° C. minute.

4. A process for pasteurizing microbial cells, the process characterized in that it comprises heating the cells and maintaining the cells at an elevated temperature (T, ° C) for a time (t, minutes) in a plateau stage where the product tT is from 140 to 100,800 ° C. minute. A process according to claim 2 or 4 characterized in that: (a) the plateau is the maximum temperature; (b) the shape of the graph of the pasteurization protocol of time (t) vs temperature (T) is a trapezoid; (c) heating and / or cooling is linear; and / or (d) the cells are heated from an initial temperature below 40 ° C and / or heated to a temperature above 70 ° C; (e) the cells contain, or produce, a PUFA or a microbial oil (which optionally contains PUFA). A process according to any of the preceding claims characterized in that the microbial cells are heated from 40 ° C to 70 ° C in no more than 15 minutes and / or the cells are heated at a speed of at least 0.6 or 1.0 ° C / minute A process according to any of the preceding claims characterized in that: (a) the microbial cells are heated at a rate of at least 2 ° C / minute; (b) the pasteurization temperature (plateau) is from 70 to 100 ° C, optimally from 70 to 85 ° C; (c) the cells are cooled at a rate of at least -0.6 or -1.6 ° C / minute, and / or (d) the area below the graph time (minute) versus temperature (° C) is less than 10,000 or 8,000 ° C. minute. A process for obtaining a PUFA or microbial cell microbial oil, the process characterized in that it comprises the pasteurization of the cells according to any of the preceding claims and the extraction or isolation of a PUFA or a microbial oil from the pasteurized cells . A microbial oil having a triguceride content of at least 90%, a peroxide index (POV) of less than 1.5 (or 1.0) and / or an anisidine index (AnV) less than 15, optionally less than 12. An oil according to claim 9 characterized in that: (a) the PUFA contains a fatty acid? -3 or (b) the PUFA content is at least 40%; (c) the PUFA contains arachidonic acid (AA), eicosapentaenoic acid (EPA) and / or docosahexaenoic acid (DHA), and / or (d) the oil is crude or unrefined.